Gbr22 'Purple' Protein Expression, Purification, and Characterization
Introduction: Recombinant proteins are proteins that are produced by introducing the DNA of the desired protein to an existing organism’s DNA. These proteins are often used in biological and biomedical sciences and must be expressed in large quantities and highly purified to use in labs. However, there exist tens of thousands of different proteins, many of which have different procedures for the optimal expression and purification of the protein. Common variables to consider include the type of cell used to express the protein, the type of vector used to carry the protein DNA, whether the protein should be tagged, and the purification strategy used.
The types of cells that could be used include bacteria, virus, yeast, insect, or human. The most common host cells are E. coli bacteria because a large number of proteins can be successfully produced in this type of cell. Trials show that about 50% of proteins from Eubacteria or Archaea and about 10% of proteins from Eukarya can be expressed in E. coli. The widespread use of E. coli can also be attributed to its fast and inexpensive nature. The particular strain of E. coli used is the BL21(DE3) strain, which is useful for high-level protein production purposes. Many proteins are also produced with an affinity tag because they aid the protein purification process. A common tag used is an N-terminus hexahistidine tag because they rarely affect the characteristics of the protein, the protein tagged can then be easily purified, and the tag is relatively small so it does not affect the solubility of the protein.
The protein expression process begins with the cloning and introduction of the protein DNA to a specified vector. Then the protein’s DNA is introduced to host cells. Next a starter culture is grown overnight before it is then used to inoculate a larger culture to express the protein. During the purification process, the cell wall is first broken to release the soluble proteins. Then the insoluble cell debris is removed and the soluble portion is filtered using the affinity tag and an elution compound leaving only the purified protein. In this lab, the BL21(DE3) strain of E. coli was used to express the gbr22 protein originally produced in the organism Montipora efflorescens. Then the protein is purified using a C-terminus hexahistidine tag along with a Ni-NTA resin. Finally the purified protein is analyzed using gel electrophoresis to test the purity of the final protein. Protein expression and purification is important because the protein taken from those experiments can then be used in x-ray crystallography, enzyme inhibition experiments, or other biological experiments.
Materials and Methods: The first portion of the lab was bacterial protein expression. First, the plasmid containing the gbr22 protein DNA was introduced to the bacteria through icing and heat shock before growing a starter culture of bacteria. Bacteria containing the gbr22 protein DNA were placed onto an Agar plate with ampicillin and bacteria without the plasmid DNA were also placed onto an Agar plate with ampicillin as control. A fun plate containing water from a nearby fish tank was also placed onto a third Agar plate without ampicillin. The three plates were then placed in a 37°C incubator overnight. Then, the plate containing the bacteria with the plasmid was used to grow a larger culture of bacteria which was then harvested by centrifuging the cells out of solution, resuspending the cells in a 1xPBS solution, and adding lysozyme to break apart the cell walls.
The second portion of the lab was protein purification. First the cells were incubated in order to ensure complete lysis of the cell. Then benzonase was added to the solution in order to digest all the DNA/RNA. After incubating the solution again, it was then centrifuged to separate the soluble portions of the cell from the insoluble portions. The solution was then filtered to remove the large molecules and the remaining solution was then put through Ni-NTA affinity purification. The six histidine amino acids prevented the gbr22 protein from being washed out in the first two rinses and once the other proteins were washed out, a 250mM solution of imidazole was used to wash out the gbr22 protein. Finally a UV-Vis spectroscopy was used to determine the concentration of the final purified protein.
The third portion of the lab was protein characterization. Using the six samples taken from the two previous portions, a SDS-PAGE gel electrophoresis was done to determine the purity of the final protein. The gel was then stained using an imperial protein stain and dried on a Whatman filter paper to view the amount of protein in each of the six samples.
Results: Protein ExpressionLab
Figure 1: Control LB Plate with AMP with no E. Coli bacteria growing. Bacteria was not injected with DNA.
Figure 2: LB plate with AMP and purple colonies of E. Coli bacteria growing. Bacteria were injected with pGEM-gbr22.
Figure 3: LB plate without antibiotics growing bacteria taken from the fish tank.
Figure 4: Large Purple E. Coli bacteria culture in a flask with LB and AMP.
Figure 5: Wet pellets of purple bacteria cells after centrifuging and decanting the liquid. Wet cell pellet weight: #1) 0.31g #2) 0.35g
Protein Purification Lab
Figure 6: The top tube contains Elution 1 which is darker than Elution 2 on the bottom because it contains most of the purple protein.
Figure 7: Trial #1 Absorbance Spectrum at 280nm wavelength for elution 1.
Figure 8: Trial #2 Absorbance Spectrum at 280nm wavelength of elution 1.
Figure 9: Trial #1 Absorbance Spectrum of elution 1 at the maximum wavelength of 574nm.
Figure 10: Trial #2 Absorbance Spectrum of elution 1 at the maximum wavelength of 574nm.
Beer's Law Calcuations: At 280nm Wavelength: A=Ebc 0.233=(38850 L/mole-cm)(1 cm) x x=0.1547 mg/mL
At Maximum Wavelength (574nm): A=Ebc 0.525=(118300 L/mole-cm)(1cm) x x=0.1145 mg/mL
Yield: At 280nm Wavelength: (0.1547 mg/mL)(5 mL)=0.7735 mg
At Maximum Wavelength (574nm): (0.1145 mg/mL)(5 mL)=0.5725 mg
Protein Characterization Lab
Figure 11: Stained, dried, and vacuumed SDS-PAGE gel. Wells 2 and 3 contain the ladder. Well 4 contains sample 1. Well 5 contains sample 2. Well 6 contains sample 3. Well 7 contains sample 4. Well 8 contains sample 5 (elution 1). Well 9 contains sample 6 (elution 2).
Discussion:
The starter culture plates appeared to have been successful because the plate with bacteria that contained DNA grew and they expressed the purple color that was indicative of the gbr22 protein. The plates also appeared uncontaminated because the control plate did not grow any bacteria. The larger culture grown in the flask also appeared to have been successful since the purple color was expressed in the flask and the wet cell pellets revealed that the cells were successfully harvested.
Looking at the Beer’s Law calculations from the protein characterization portion of the lab, there is a 0.0402 mg/mL difference between the maximum wavelength concentration and the 280nm wavelength concentration. The yield calculations also reflect this difference. This difference in concentration could be due to either an impure elution 1 or errors in the UV-VIS spectroscopy.
After comparing the molecular weight standard to the elution 1 results in lane 8 the molecular weight of the gbr22 protein appears to be slightly less than 25kDa. Elution 1 also appears to have a few impurities since there are about 3 bands of protein that is not the purple protein. This suggests that the final purity of sample 5 which is elution 1 is roughly 90% because the other proteins in sample 5 are very faint. The error could be from contamination from other wells or simply an impure elution 1.
Conclusions:
In this lab, the protein gbr22, a purple protein from the coral Montipora efflorescens, was overexpressed in an E. coli bacteria cell. The protein was then purified and six samples were taken during the purification process. The samples were then analyzed using gel electrophoresis to determine the final protein’s purity. The next step would be to take the protein and test how well cer
Figure 12: Fermentas PageRuler Prestained Protein Ladder.
tain ligands bind to it in the wet lab.
References: 1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods2008, 5 (2), 135-46
Gbr22 'Purple' Protein Expression, Purification, and Characterization
Introduction:Recombinant proteins are proteins that are produced by introducing the DNA of the desired protein to an existing organism’s DNA. These proteins are often used in biological and biomedical sciences and must be expressed in large quantities and highly purified to use in labs. However, there exist tens of thousands of different proteins, many of which have different procedures for the optimal expression and purification of the protein. Common variables to consider include the type of cell used to express the protein, the type of vector used to carry the protein DNA, whether the protein should be tagged, and the purification strategy used.
The types of cells that could be used include bacteria, virus, yeast, insect, or human. The most common host cells are E. coli bacteria because a large number of proteins can be successfully produced in this type of cell. Trials show that about 50% of proteins from Eubacteria or Archaea and about 10% of proteins from Eukarya can be expressed in E. coli. The widespread use of E. coli can also be attributed to its fast and inexpensive nature. The particular strain of E. coli used is the BL21(DE3) strain, which is useful for high-level protein production purposes. Many proteins are also produced with an affinity tag because they aid the protein purification process. A common tag used is an N-terminus hexahistidine tag because they rarely affect the characteristics of the protein, the protein tagged can then be easily purified, and the tag is relatively small so it does not affect the solubility of the protein.
The protein expression process begins with the cloning and introduction of the protein DNA to a specified vector. Then the protein’s DNA is introduced to host cells. Next a starter culture is grown overnight before it is then used to inoculate a larger culture to express the protein. During the purification process, the cell wall is first broken to release the soluble proteins. Then the insoluble cell debris is removed and the soluble portion is filtered using the affinity tag and an elution compound leaving only the purified protein.
In this lab, the BL21(DE3) strain of E. coli was used to express the gbr22 protein originally produced in the organism Montipora efflorescens. Then the protein is purified using a C-terminus hexahistidine tag along with a Ni-NTA resin. Finally the purified protein is analyzed using gel electrophoresis to test the purity of the final protein. Protein expression and purification is important because the protein taken from those experiments can then be used in x-ray crystallography, enzyme inhibition experiments, or other biological experiments.
Materials and Methods:
The first portion of the lab was bacterial protein expression. First, the plasmid containing the gbr22 protein DNA was introduced to the bacteria through icing and heat shock before growing a starter culture of bacteria. Bacteria containing the gbr22 protein DNA were placed onto an Agar plate with ampicillin and bacteria without the plasmid DNA were also placed onto an Agar plate with ampicillin as control. A fun plate containing water from a nearby fish tank was also placed onto a third Agar plate without ampicillin. The three plates were then placed in a 37°C incubator overnight. Then, the plate containing the bacteria with the plasmid was used to grow a larger culture of bacteria which was then harvested by centrifuging the cells out of solution, resuspending the cells in a 1xPBS solution, and adding lysozyme to break apart the cell walls.
The second portion of the lab was protein purification. First the cells were incubated in order to ensure complete lysis of the cell. Then benzonase was added to the solution in order to digest all the DNA/RNA. After incubating the solution again, it was then centrifuged to separate the soluble portions of the cell from the insoluble portions. The solution was then filtered to remove the large molecules and the remaining solution was then put through Ni-NTA affinity purification. The six histidine amino acids prevented the gbr22 protein from being washed out in the first two rinses and once the other proteins were washed out, a 250mM solution of imidazole was used to wash out the gbr22 protein. Finally a UV-Vis spectroscopy was used to determine the concentration of the final purified protein.
The third portion of the lab was protein characterization. Using the six samples taken from the two previous portions, a SDS-PAGE gel electrophoresis was done to determine the purity of the final protein. The gel was then stained using an imperial protein stain and dried on a Whatman filter paper to view the amount of protein in each of the six samples.
Results:
Protein Expression Lab
Protein Purification Lab
Beer's Law Calcuations:
At 280nm Wavelength:
A=Ebc
0.233=(38850 L/mole-cm)(1 cm) x
x=0.1547 mg/mL
At Maximum Wavelength (574nm):
A=Ebc
0.525=(118300 L/mole-cm)(1cm) x
x=0.1145 mg/mL
Yield:
At 280nm Wavelength:
(0.1547 mg/mL)(5 mL)=0.7735 mg
At Maximum Wavelength (574nm):
(0.1145 mg/mL)(5 mL)=0.5725 mg
Protein Characterization Lab
Discussion:
The starter culture plates appeared to have been successful because the plate with bacteria that contained DNA grew and they expressed the purple color that was indicative of the gbr22 protein. The plates also appeared uncontaminated because the control plate did not grow any bacteria. The larger culture grown in the flask also appeared to have been successful since the purple color was expressed in the flask and the wet cell pellets revealed that the cells were successfully harvested.
Looking at the Beer’s Law calculations from the protein characterization portion of the lab, there is a 0.0402 mg/mL difference between the maximum wavelength concentration and the 280nm wavelength concentration. The yield calculations also reflect this difference. This difference in concentration could be due to either an impure elution 1 or errors in the UV-VIS spectroscopy.
After comparing the molecular weight standard to the elution 1 results in lane 8 the molecular weight of the gbr22 protein appears to be slightly less than 25kDa. Elution 1 also appears to have a few impurities since there are about 3 bands of protein that is not the purple protein. This suggests that the final purity of sample 5 which is elution 1 is roughly 90% because the other proteins in sample 5 are very faint. The error could be from contamination from other wells or simply an impure elution 1.
Conclusions:
In this lab, the protein gbr22, a purple protein from the coral Montipora efflorescens, was overexpressed in an E. coli bacteria cell. The protein was then purified and six samples were taken during the purification process. The samples were then analyzed using gel electrophoresis to determine the final protein’s purity. The next step would be to take the
protein and test how well cer
References:
1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46
2) Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/ (accessed April 18, 2011)